Biology Reference
In-Depth Information
visualize the patterns under the microscope. The proteins will bind tightly to the
UV-exposed PEG-PLL regions, serving as substrates of cell adhesion.
2.
Put the coverslips into 0% FBS medium in wells in an incubator for 5 min before
seeding the cells. High concentrations of serum might impair attaching and
spreading.
3.
Wash, in PBS, adherent cells that have been growing for a couple of days
undisturbed. Detach the cells with trypsin-EDTA at 37
C for 3-10 min.
4.
Carefully but resolutely resuspend the cells in complete medium, for example, by
pressing the pipette tip against the bottom of the flask while dispensing them. Use
a total of 5 ml for a T25 flask. Add cells to the wells with the coverslips (e.g.,
400
l cell suspension per 3.5 cm well). Alternatively, remove an aliquot for
counting while spinning the rest. Seed the cells on the coverslips at a density of
approximately 10,000 cells/cm
2
.
5.
Put the coverslips into the incubator for a brief time (10-20 min for RPE1 cells)
before checking the cells. When the cells are attached, ideally there is one cell
stuck to each pattern, and the pattern array is easily visible. Wash away
unattached excess cells gently. It is important to never remove all medium, since
the cells would quickly dry out due to the hydrophobicity of the PEG-PLL
coating on the coverslip. HEPES can be used for better buffering outside of the
incubator for steps taking longer time, like the washing step.
6.
Let the cells spread fully in the incubator (2-4 h for RPE1 cells). Cells are now
ready to be visualized live or alternatively be fixed and stained for later
visualization. Cells can be fixed with PAF by adding one volume of 6% PAF in
PBS to coverslips placed in one volume of medium. If the mitochondria are to be
visualized using MitoTracker, incubation (15-30 min) with MitoTracker must
take place before fixing.
m
7.4
MITOCHONDRIA AND MICROTUBULE VISUALIZATION
We typically use RPE1 cells expressing fluorescent markers for mitochondria. There
are many protocols for labeling cells with the fluorescent marker of interest. We
briefly describe below the methods we use.
7.4.1
Mitochondria and microtubule labeling
There are several ways to fluorescently label the mitochondria and microtubules. For
fixed cells, there are a multitude of antibodies against different mitochondrial pro-
teins, and a multitude of antibodies against microtubules. A general method for fix-
ing and staining cells follows:
1.
Place the coverslips cell side up in drops of PBS in a humid chamber.
2.
Wash the coverslips containing the spread cells in micropatterns with PBS, by
alternately aspiring and pipetting 1 ml of PBS, carefully to prevent dehydration.
